1. Field of the Invention
This invention generally relates to methods, storage mediums, and systems for analyzing particle quantity and distribution within an imaging region of an assay analysis system and, further, to methods, storage mediums, and systems for evaluating the performance of a focusing routine performed on an assay analysis system.
2. Description of the Related Art
The following descriptions and examples are not admitted to be prior art by virtue of their inclusion within this section.
Fluid assays are used for a variety of purposes, including but not limited to biological screenings and environmental assessments. Often, particles are used in fluid assays to aid in the detection and quantification of one or more analytes of interest within a sample. In particular, particles provide substrates for carrying reagents configured to react with analytes of interest within a sample such that the analytes may be detected. In some cases, a multiplexing scheme is employed in assay analysis systems such that multiple analytes may be evaluated in a single analysis process for a single sample. To facilitate a multiplexing scheme, particles are configured into distinguishable groups and each group is used to indicate the presence, absence, and/or amount of a different analyte in an assay. The different particle subsets may be distinguishable, for example, by different fluorescent dyes and/or different concentrations of dyes absorbed into particles and/or bound to the surface of particles. In addition or alternatively, the size of particles among the different subsets may vary. In any case, optical imaging instruments may be used to analyze fluid assays induced with particles. More specifically, optical imaging instruments may be configured to image particles within an illuminated region of a chamber in which an assay is introduced and may be further configured to analyze the imaged particles for the detection and quantification of one or more analytes of interest.
It is often advantageous to monitor the quantity of particles introduced into the chamber to insure an appropriate amount is in the imaging region for analysis of the sample. In particular, if an imaging region is overpopulated with particles, at least some of the particles will crowd each other causing them to reflect each other's light and falsely convey a brighter intensity. On the contrary, if the number of particles within an imaging region is not enough to constitute statistically significant data regarding the detection and quantification of analytes of interest within a sample (which may be particularly applicable in multiplexing schemes), then the processes of imaging the particles and processing the data acquired therefrom are performed in vain. In some cases, the number of particles within the imaging region (particularly when there is not enough particles within the imaging region) may affect the accuracy of an autofocus routine performed on the imaging system, which in turn will affect the resolution of any image taken and consequently skew data acquired therefrom.
In many instances, the number of particles delivered into the imaging chamber is estimated based on expected particle densities present in the sample volume. Particle densities, however, can vary substantially from sample to sample and, thus, such a technique requires the particle density of a sample to be known and inputted into a system prior to injecting the sample into the imaging chamber. Some assay analysis techniques involve counting particles within an imaging chamber. For example, spatial-domain image analysis is often performed using thresholding for edge or peak detection of particles. Thresholding, however, can become complicated if particle brightness varies significantly (such as in a multiplexing scheme). Furthermore, once an edge or peak has been detected, a neighborhood of pixels must be assembled that identifies an imaged particle, which can sometimes be time-consuming. Thus, spatial-domain image analysis is not generally considered advantageous for monitoring the quantity of particles introduced into a chamber.
In an optical imaging instrument that must accurately measure the amount of fluorescent light emitted from each observed particle, particle distribution within an imaging region is as important as quantity. In particular, similar to the overpopulation of particles within an imaging region, particles that are clustered together may induce measurable reflections and falsely convey a brighter intensity. Moreover, light collected from a cluster of particles is generally difficult to differentiate on a particle-by-particle basis. To overcome this problem, particle clusters will often be ignored during analysis. Therefore, two imaging volumes containing an equal population of particles but different distributions will yield different amounts of useful data. In order to identify the particle clusters, spatial-domain image analysis as described above for particle counting is often performed. Such a technique, however, is generally not used to determine particle distribution in an imaging volume nor would it be considered a viable option, particularly as particles are being introduced into an imaging chamber, due to its time constraints.
In addition to the number of particles arranged within an imaging region of an optical assay analysis system, the configuration of the system affects the accuracy of the data obtained from an image. In particular, it is important that the focal position of the photosensitive detection subsystem is optimized such that image resolution is optimized and accurate data is obtained. In light of its importance, many optical analysis systems employ an automated routine for periodically optimizing the focal position of its photosensitive detection subsystem. In many cases, however, characteristics and/or operation of an optical analysis system may change over time and, in some embodiments, the changes may affect a routine's ability to optimize a focal position of a photosensitive detection subsystem.
Accordingly, it would be beneficial to develop methods, program instructions, and systems for evaluating the performance of a focusing routine performed on an optical assay analysis system. Furthermore, it would be desirable to develop methods, program instructions, and systems for analyzing particle quantity and distribution within an imaging region of an optical assay analysis system, particularly as particles are delivered into the imaging region. More specifically, it would be advantageous to develop methods, program instructions, and systems for analyzing the quantity of particles within an imaging chamber to insure an appropriate amount is present for further processes conducted by the system.